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Ovary |
Laboratory of Animal Reproduction, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-amamiyamachi, Aoba-ku, Sendai 981-8555, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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follicular development, granulosa cells, growth factors, ovary, theca cells
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In general, angiogenesis is mediated via vascular endothelial growth factor (VEGF), which plays an important role in the process of fetal and neonatal development [6–8]. Also known as vascular permeability factor, VEGF was originally isolated because of its specific ability to stimulate microvascular endothelial cells to proliferate and migrate as well as to enhance vascular permeability [9, 10]. The human VEGF gene produces five isoforms of proteins, with 121, 145, 165, 189, and 206 amino acids (VEGF 121–206), by alternative splicing of the VEGF mRNA [6, 11]. Porcine VEGF is shorter by one amino acid compared to human VEGF, and it has a potential glycosylation site at Asn-74 [12]. The VEGF acts via two tyrosine kinase-family receptors, namely Flt-1 (VEGFR-1) and Flk-1/KDR (VEGFR-2) [9, 13]. Experimental research in which systemic endocrine changes were precisely designed and controlled indicates that VEGF participates in the development of blood vessels in the thecal layer of follicles [14–16]. Moreover, treatment with anti-VEGF (VEGF TrapA40) to severely restrict follicular angiogenesis during follicular development reduced angiogenesis in the endothelial cell area of the thecal cell layer and produced a marked decline in Flt-1 mRNA expression, resulting in an 87% decrease in proliferation in the theca of the antral follicles [17, 18]. A VEGF antagonist, VEGF Trap R1R2, inhibited the thecal vasculature for follicular development throughout the follicular phase of the cycle in a primate [19]. Therefore, VEGF is thought to be a central factor in regulating thecal angiogenesis during follicular development. Moreover, local administration of angiogenic inhibitors (TNP-470 or angiostatin) or soluble VEGFR-1 (VEGF antagonist) into the preovulatory follicle of rhesus monkeys during a spontaneous menstrual cycle indicated that VEGF antagonist impairs ovulation and attenuates subsequent luteal function, possibly by altering normal steroidogenic-nonsteroidogenic cell interaction or differentiation without dramatically changing cell numbers [20, 21]. In turn, these findings may raise the possibility that enhancement of the circular commitment of VEGF around the follicles could positively affect their developmental process (follicle selection). We tested this hypothesis by inducing extraproduction of VEGF by direct ovarian injection of its gene fragments in pigs.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Using a TAP Express Kit (Gene Therapy Systems, Inc., San Diego, CA), porcine VEGF gene fragments (product size: VEGF 120, 1357 base pairs [bp]; VEGF 164, 1489 bp) were transformed into a transcriptionally active polymerase chain reaction (PCR) fragment to be used for direct introduction into mammalian tissues according to the manufacturer's instructions. Briefly, custom oligos were used to produce the TAP primary fragment, which was then added to TAP Express promoter and terminator mixes. The 5'-custom and 3'-custom oligo must contain 46 and 40 nucleotides (nt), respectively; of these, 26 nt (5'-CTG CAG GCA CCG TCG TCG ACT TAA CA-3') and 20 (nt 5'-CAT CAA TGT ATC TTA TCA TGT CTG A-3') comprise the 5'- and 3'-TAP Ends, respectively. Universal sequence and the other 20 nt (VEGF sequence: 5'-ATG AAC TTT CTG CTG TCT TG-3' and 5'-TCA CCG CCT CGG CTT GTC AC-3', GenBank accession no. X81380) are used to make up the porcine VEGF sequence. In the preliminary examination, we confirmed that the green fluorescent protein gene provided in the kit was expressed in porcine fibroblasts. To investigate the effect of VEGF gene fragment injection on follicular development, 11 prepubertal miniature pigs (CSK Research Park, Inc., Suwa, Japan) at 3 mo of age and a body weight of 9.2–11.8 kg were used and divided in three groups. The first group (n = 4) was injected i.m. with 500 IU of eCG (Teikoku Zouki Pharmaceutical Co., Tokyo, Japan) to induce follicular development, and the second was directly injected with saline as a control (n = 4) into the medulla of both ovaries with a 30-gauge needle. The third group (n = 3) was directly injected with VEGF gene fragments (10 µg DNA/ovary) into the medulla of both ovaries, followed by administration i.m. of 500 IU of eCG 7 days later to assess the effect of the injected VEGF gene fragments. The VEGF gene fragments (10 µg per 10 µl) were added to 10 µl of GenePORTER reagent (Gene Therapy Systems, Inc., San Diego, CA). After anesthetization by injection of ketamine hydrochloride (5 ml/gilt; Sankyo Co. Ltd., Tokyo, Japan) and atropine sodium salt (0.5 mg/gilt; Tanabe Co. Ltd., Tokyo, Japan), the animals from each group were ovariectomized 72 h [22–24] after eCG or saline injection to examine the follicular population (right ovaries) and to detect mRNA expression (left ovaries). Both ovaries removed from individual animals of each group had the same number of follicles protruding on the ovarian surface. The present study was approved by the Ethics Committee for Care and Use of Laboratory Animals for Biomedical Research of the Graduate School of Agricultural Science, Tohoku University.
Histological Examination
The right ovaries were fixed in 4% paraformaldehyde solution, embedded in paraffin wax, and then sectioned serially (thickness, 8 µm). Every 10th section was mounted and stained with hematoxylin-eosin. All antral follicles larger than 0.5 mm in diameter were counted in individual sections. To avoid counting the same follicle more than once, only individual follicles having an oocyte with a nucleus were evaluated, and the size of the follicle in which the oocyte was present was measured using an ocular micrometer. Each follicle was classified as either healthy or atretic by the absence or presence of 10 pyknotic bodies in the granulosa cells of the section, respectively. On the basis of their diameter, the follicles were categorized into four classes: 0.5–0.9, 1.0–2.9, 3.0–3.9, and 
4.0 mm. In addition, to assess capillary density, the follicles greater than 3.0 mm in diameter were recategorized into four classes: 3.0–3.9, 4.0–4.9, 5.0–6.0, and >6.0 mm. All capillaries larger than 10 µm in diameter in the section of whole theca interna of follicles larger than 3.0 mm in diameter were counted under 200x magnification (20x objective lens and 10x ocular lens). Twenty-nine, 28, 19, and 7 follicles of 3.0–3.9, 4.0–4.9, 5.0–6.0, and >6.0 mm in diameter, respectively, were used to assess the capillary density in the theca interna.
Isolation of Granulosa Cells and Follicular Fluids
The follicles (eCG alone, n = 5; eCG plus VEGF, n = 5) were isolated from the left ovaries of each animal as previously described [16]. Briefly, the individual follicles were isolated in dissection medium (Dulbecco phosphate-buffered saline supplemented with 0.4% BSA) and were directly transferred to 1 ml of dissection medium to mechanically separate the granulosa cells and follicular fluid from the thecal shells by gently scraping the follicles with a pair of fine forceps. The medium containing dispersed granulosa cells and follicular fluid was collected and centrifuged. Supernatant was diluted 10-fold with PBS as diluted follicular fluid. The thecal shells obtained were then vigorously vortexed and carefully washed to remove any possible contamination from the granulosa cells. The granulosa cells, thecal shells, and diluted follicular fluids were then stored in liquid nitrogen for analysis of mRNA expressions of VEGF 120 and 164, mRNA expressions of Flt-1, and Flk-1, and concentration of VEGF, respectively.
Expression of VEGF 120, VEGF 164, Flt-1, and Flk-1 mRNAs
Total cellular RNA was extracted from the granulosa and thecal tissues with the RNeasy Mini Kit (Qiagen K.K., Tokyo, Japan). Semiquantitative reverse transcription (RT)-PCR was performed using Ready To Go RT-PCR Beads (Amersham Pharmacia Biotech, Inc., Piscataway, NJ) following the method provided by the manufacturer with 100 ng of RNA. The RT reaction was carried out at 42°C for 15 min, and the samples were incubated at 95°C for 5 min to inactivate the reverse transcriptase and to completely denature the template. Primers for VEGF (GenBank accession no. X81380) were 5'-CCT GAT GCG GTG CGG GGG CT-3' (nt 779–798) and 5'-TGG TGG TGG CGG CGG CTA TG-3' (nt 1197–1216). Because these primers were designed to initiate before and to terminate after the splicing site, they enable amplification of all VEGF isoforms. Within the ovary, VEGF 120 and VEGF 164 are the prevalent isoforms [25] and are the variants measured in follicular fluid. Primers for Flt-1 (GenBank accession no. AJ245445) were 5'-AGA GCG ACG TGT GGT CCT AC-3' (nt 6–25) and 5'-AGT CTT TGC CGT CCT GTT GT-3' (nt 251–270). Primers for Flk-1 (GenBank accession no. AF233076) were 5'-TTG TCG GAA AAG AAC GTG GT-3' (nt 75–94) and 5'-TGC CAT CCT GTT GAG CAT TA-3' (nt 467–486). Primers for ß-actin [14, 15] were 5'-ATC GTG CGG GAC ATC AAG GA-3' (ActSS-1) and 5'-AGG AGG GAG GGC TGG AAG AG-3' (ActSS-2). The amplification profiles of each gene are shown in Table 1. The final cycle included a further 5 min at 72°C for complete strand extension. The RT-PCR products were electrophoresed on a 2% agarose gel and visualized by ethidium bromide staining. The bands were quantified by densitometry using the NIH Image 1.63 analysis program (National Institutes of Health, Bethesda, MD). ß-Actin mRNA has been found in pig follicle cells with levels that are independent of follicle status and size [26], and its expression is not affected by growth factors or gonadotropins [27, 28]. Therefore, each gene mRNA level in the present study was normalized on the basis of ß-actin mRNA content.
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Concentration of VEGF
The samples obtained from diluted follicular fluids of single follicles were measured for their VEGF content using a specific ELISA (Quantikine; R&D Systems, Minneapolis, MN) that has been validated for the measurement of porcine VEGF [14]. A 96-well plate reader set to read at an emission of 450 nm was used to quantify the contents. The sensitivity of the assay was 0.1 ng/ml, and the intra- and interassay coefficients of variations were 5.7% and 7.3%, respectively. The level of VEGF in the samples of single follicular fluids was expressed as ng/ml follicular fluid.
Statistical Analysis
All data are presented as the mean ± SEM. Significant differences in the ovarian weight and the number of follicles per ovary among the control, eCG-alone, and VEGF-plus-eCG groups were analyzed by the Kruskal-Wallis nonparametric test. The capillary density in the thecal cell layer among the control, eCG-alone, and VEGF-plus-eCG groups was analyzed by ANOVA, followed by the Fisher Least Significant Difference test as a multiple-comparison test. The percentage of atretic follicles at the different developmental stages was analyzed by the chi-square test. The mRNA levels of VEGF 120, VEGF 164, Flt-1, and Flk-1 were normalized on the basis of ß-actin mRNA content, and significant differences in each of the genes between the eCG-alone and VEGF-plus-eCG groups were analyzed by the Student t-test. Differences were considered to be significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In prepubertal gilts, treatment with exogenous gonadotropins, such as eCG and hCG, can induce follicular development [29]. Large follicles (diameter, >5 mm) protruding on the ovarian surface were observed in the ovaries of eCG-treated gilts with or without VEGF gene fragment injection, but the mean number of large follicles significantly increased (P < 0.05) in the VEGF gene fragment-treated ovaries (14.8 ± 1.89 vs. 8.3 ± 0.75 per ovary) (Fig. 1A). In the VEGF gene fragment-treated ovaries in particular, the large follicles protruding from the surface of the ovary were rich in visible blood vessels with erythrocytes. The average weight of the VEGF gene fragment-treated ovaries was significantly greater (P < 0.05) than those of the control and the eCG-alone groups (Fig. 1B). Preovulatory follicles larger than 4 mm in diameter were observed in the eCG-treated ovaries with or without VEGF gene fragment injection. The VEGF gene fragment injection resulted in the appearance of a large follicle of 9.0 mm in diameter. The number of preovulatory follicles increased in the VEGF gene fragment-treated ovaries compared with those of either the control or the eCG-alone group (Figs. 1A and 2A). The VEGF gene fragment injection did not affect the number of follicles of 3.0–3.9 mm in diameter, whereas the number of follicles of 1.0–2.9 mm in diameter decreased (P < 0.05) in the VEGF gene fragment-treated ovaries compared with the control. Although the percentage of atretic follicles of 1.0–2.9 mm in diameter increased (P < 0.05) in the eCG-treated ovaries with or without VEGF gene fragment injection compared with the control, no atretic follicle larger than 3.0 mm was observed in the ovaries treated with VEGF gene fragment injection (Fig. 2B).
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Promotion of Perifollicular Angiogenesis by VEGF Gene Fragment Injection
To analyze the effect of the VEGF gene fragment injection on the development of perifollicular angiogenesis, we investigated the vascular density in the theca interna using serial sections of the ovaries. Histological examination revealed that the VEGF gene fragment injection markedly increased (P < 0.05) the vascular density (number/mm2 theca interna area) in the theca interna of the follicles larger than 3.0 mm in diameter (Fig. 3). In the ovaries injected with VEGF gene fragment, the vascular density in the follicles ranged between 3.0 and 4.9 mm in diameter, showing an increase of approximately 2-fold compared with those of the eCG-alone group (P < 0.05). Follicles larger than 6.0 mm in diameter, which appeared only in the gilts that received the injection of VEGF gene fragments, had significantly higher (P < 0.05) vascular density than in other follicles.
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Extraexpression of VEGF During the Follicle Growth Phase
We previously reported that VEGF 120 and VEGF 164 are important mediators of follicular angiogenesis during porcine follicular development [16]. In the present study, we performed direct injection of VEGF gene fragments into miniature porcine ovaries to promote perifollicular angiogenesis. Semiquantitative RT-PCR showed that VEGF 120 and VEGF 164 mRNAs were greatly expressed in granulosa cells of the follicles in the VEGF gene fragment-treated ovaries (Fig. 4A). Because VEGF 120 and VEGF 164 are freely soluble proteins, we measured the concentration of VEGF in the follicular fluid using an ELISA. The VEGF concentration was significantly higher (P < 0.05) in the follicular fluid derived from the follicles of the ovaries injected with the VEGF gene fragments (140.2 ± 41.2 ng/ml) than in those injected with eCG only (37.8 ± 11.2 ng/ml) (Fig. 4B).
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Expression of VEGF Receptors
The expression of Flt-1 mRNA appeared to be increased in the thecal cell layers of the follicles from VEGF-treated ovaries (Fig. 5). However, because of a high SEM, this apparent increase was not statistically significant. The expression of Flk-1 mRNA was unaffected by the VEGF gene fragment injection (Fig. 5).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Histological examination revealed that the vascular density in the theca interna of follicles in the VEGF-treated ovary increased 2-fold compared with that in the VEGF-untreated ovary. The pattern of perifollicular angiogenesis in the treated group was essentially the same during follicular development after eCG treatment. These changes clearly indicated stimulatory signs of thecal angiogenesis associated with a significant amount of relative tissue areas covered by vessels. Because perifollicular angiogenesis supplies gonadotropins and many growth factors to the granulosa cells of follicles, the progress in perifollicular angiogenesis is associated with the promotion of follicular development. In the present study, our findings demonstrate that direct single injection of VEGF gene fragments into the ovary promoted perifollicular vascularization and a resultant increase in the size and number of follicles. Thus, our data support the concept that follicle growth is associated with and, perhaps, is critically dependent on the degree of vascular development [4].
Importantly, the increase in vascular density surrounding the antral follicles contributes to the inhibition of atresia. Early atretic follicles can regenerate when placed in culture, suggesting that the follicle remains in the atretic state because of a decrease in vascularity that limits access to nutrients, substrates, and trophic hormones [30]. Our findings demonstrated that promotion of thecal vascularization by VEGF contributed to the decrease in atretic follicles. In pig ovaries, as in those of other species, apoptotic cell death is a mechanism by which follicular atresia is induced [31]. The VEGF reduces tumor cell apoptosis, whereas inhibition with anti-VEGF neutralizing antibodies induces apoptosis directly in tumor cells [32]. Thus, in addition to its roles in angiogenesis and vessel permeability, VEGF may act as a survival factor for the granulosa cells of follicles and may then suppress atresia of the antral follicles, leading to an increased number of ovulated oocytes.
Because VEGF acts via two tyrosine kinase-family receptors, namely Flt-1 and Flk-1 [9, 13], we analyzed the mRNA expression of VEGF receptors in the theca cells when VEGF mRNA was extraexpressed in the granulosa cells. The VEGF binds to its receptors, such as Flt-1, Flt-4, Flk-1, and neuropilin-1, in the vascular endothelial cells [33–35]. Of these receptors, Flt-1 and Flk-1 both bind to VEGF with high affinity [9]. Although the expressions of Flt-1 and Flk-1 mRNAs were detected in the ovary [16, 36], it is still unknown whether Flt-1 or Flk-1 is predominantly committed to thecal angiogenesis. Our results showed that the expression of Flt-1 mRNA increased more than the expression of Flk-1 in the theca cells when VEGF in the granulosa cells was extraexpressed, indicating that Flt-1 may be involved predominantly in the development of the capillary network in the theca interna during follicular development.
In conclusion, our findings demonstrated that direct injection of the VEGF gene fragments into the ovary results in development of the vascular network in the thecal cell layers and can promote the resultant follicular development. The oocytes collected from preovulatory follicles of the porcine ovaries treated with VEGF gene injection were normal in fertility and cleavage when examined by in vitro fertilization (data not shown). From the present study using pigs, we conclude that gene treatment in combination with hormonal treatment can significantly increase the number of follicles with healthy oocytes by rescuing the atretic follicles. Furthermore, we suggest that these results may offer a protocol with which to develop a novel therapy for the prevention and treatment of infertility associated with ovarian dysfunction.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Takashi Shimizu, Laboratory of Animal Reproduction, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-amamiyamachi, Aoba-ku, Sendai 981-8555, Japan. FAX: 81 22 717 8687; shimizut{at}bios.tohoku.ac.jp
Received: 12 February 2003.
First decision: 5 March 2003.
Accepted: 21 May 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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